Synthesis of NQO1-activatable Optoacoustic Probe and Its Imaging of Breast Cancer

doi:10.6023/A20100459

Abstract

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a cytosolic two-electron-specific reductase whose abnormal expression is associated with breast cancer, and NQO1 has been regarded as an important biomarker for monitoring the occurrence and development of breast tumors. Although there are some research reports involving fluorescent probes for imaging NQO1 in vivo, they generally suffer from the strong light scattering by tissues which would cause the spatial resolution of fluorescent signals degrade rapidly with imaging depth. On the other hand, as an emerging detection and imaging modality, optoacoustic tomography (OAT) is capable of providing more detailed information in deep biological tissues than fluorescent imaging, since in OAT ultrasound signals are the reporting signals. To overcome the inherent defects of fluorescent imaging, in this work, we developed a novel near-infrared (NIR) NQO1-activatable optoacoustic (OA) probe TPA-X-Q based on the push-pull internal charge transfer (ICT) scaffold. TPA-X-Q consists of a NQO1-responsive moiety, quinone propionate group and a NIR chromophore TPA-X-OH. In the absence of the enzyme NQO1, the probe displays nearly no noticeable OA signal upon NIR excitation. Whereas in the presence of NQO1, the quinone propionate group of TPA-X-Q is reduced and cleaved by the enzymatic reaction triggered by NQO1, and thus TPA-X-Q is transformed into TPA-X-OH, consequently evident OA signal is generated, thereby realizing the detection and imaging of NQO1. As for the probe, the limit of detection (LOD) is determined to be 0.193 μg·mL ^–1. Furthermore, the probe TPA-X-Q displays several advantages, such as quite good selectivity towards NQO1, low cytotoxicity and excellent photostability. Moreover, the probe has been successfully utilized to detect and image the overexpressed NQO1 in the breast cancer-bearing mouse model. Orthogonal-view three-dimensional (3D) images can also be obtained by using multispectral optoacoustic tomography (MSOT), and thus precise localization of the breast cancer tumors can be achieved. This probe holds great potential for being employed as an efficacious tool for diagnosing breast cancer via responding to NQO1.

Key words: NQO1, optoacoustic probe, near-infrared, breast cancer, MSOT

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Jing Huang, Chao Wang, Mingang Lin, Fang Zeng, Shuizhu Wu. Synthesis of NQO1-activatable Optoacoustic Probe and Its Imaging of Breast Cancer[J]. Acta Chimica Sinica, 2021, 79(3): 331-337.

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Fig. & Tab. 5

Figure 1. The response and imaging mechanism of TPA-X-Q

Figure 2. (A) Absorption spectra of TPA-X-Q (10 μmol·L –1), TPA-X-OH (10 μmol·L –1), TPA-X-Q upon reaction with NQO1 (the concentration of probe was 10 μmol·L –1; that of NQO1 was 15 μg·mL –1; the reaction time was 40 min). (B) The HR mass spectrum of TPA-X-Q upon reaction with NQO1 (the concentration of probe was 10 μmol·L –1; that of NQO1 was 15 μg·mL –1; the reaction time was 10 min). (C) Time-dependent absorption spectra (0~40 min) of the probe in the presence of NQO1 (15 μg·mL –1). (D) Absorption spectra at 40 min of the probe (10 μmol·L –1) in the presence of varied levels of NQO1 (0~15 μg·mL –1). These measurements were performed in PBS (pH=7.4, containing DMSO(φ=1%) and 100 μmol·L–1 NADH cofactor) at 37 ℃

Figure 3. (A) The wavelength dependence of probe’s (10 μmol·L–1) normalized optoacoustic(OA) intensity in the presence of NQO1(15 μg·mL –1). (B) OA response of the probe (10 μmol·L –1) to NQO1 of varied levels (sample size n=3); The inset shows the representative optoacoustic images of the probe in phantom at varied NQO1 level; Excitation wavelength: 710 nm. (C) OA response of the probe (10 μmol·L –1) in the presence of NQO1(15 μg·mL –1) and (D) anti-interference test (n=3); The concentration of NaCl, KCl, alanine (Ala), arginine (Arg), tyrosine (Tyr), homocysteine (Hcy), cysteine (Cys), DL-dithiothreitol (DTT), glutathione (GSH), Urea, ascorbic acid (AA), serine was 1 mmol·L–1; that of BSA was 20 mg·mL–1; that of glucose was 10 mmol·L–1. These measurements were performed in PBS (pH=7.4, containing DMSO(φ=1%) and 100 μmol·L–1 NADH cofactor) at 37 ℃

Figure 4. (A) Representative bright-field images of nude mouse 1 week after injection of PBS (Control) or 4T1 cell suspension (Breast cancer model). (B) Representative cross-sectional OA images of the breast cancer-bearing nude mouse model before injection of the probe (0 min) and 10, 30, 60 min after injection of the probe; the color bar on the right side reflects different OA intensities. (Upper row: the OA signal image of activated probe; lower row: the overlay of the OA signal images of activated probe and grayscale background)

Figure 5. Representative 3D OA images of the breast cancer-bearing nude mouse model before injection of the probe (0 min) and 10, 30, 60 min after injection of the probe; the color bar on the right side reflects different OA intensities. (Upper row: the OA signal image of activated probe; lower row: the overlay of the OA signal images of activated probe and grayscale background)

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